Vascular endothelial growth factor A (VEGF-A), which signals through the receptor tyrosine kinases VEGFR-1 and VEGFR-2, plays a dominant role in physiologic and pathologic angiogenesis, with VEGFR-2 implicated as its principal pro-angiogenic transducer. The function of VEGFR-1 is more nebulous. Although deletion of the vegfr-1 gene results in embryonic lethality due to endothelial overcrowding (Fong, G., Zhang, L., Bryce, D. & Peng, J. Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. (1999) Development 126, 3015-3025), vascular development is grossly unscathed in mice with a deletion of its kinase domain (vegfr-1 tk−/−) (Hiratsuka, S., Minowa, O., Kuno, J., Noda, T. & Shibuya, M. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. (1998) Proc Natl Acad Sci USA 95, 9349-54), suggesting that VEGFR-1 subserves a negative role in embryogenesis by acting as a scavenger/decoy. However, conflicting data about VEGFR-1 function has emerged from studies that demonstrate that it both amplifies (Carmeliet, P. et al. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. (2001) Nat Med 7, 575-83; Hiratsuka, S. et al. Involvement of flt-1 tyrosine kinase (vascular endothelial growth factor receptor-1) in pathological angiogenesis. (2001) Cancer Res 61, 1207-1213) and antagonizes (Bussolati, B. et al. Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide. (2001) Am J Pathol 159, 993-1008) pathologic angiogenesis. Thus VEGFR-1 signaling appears to be highly cell/tissue-specific and context/stage-dependent.
Choroidal neovascularization (CNV) is the principal cause of blindness in patients with age-related macular degeneration (AMD), which is responsible for vision loss in 25-30 million people worldwide. Smith, W. et al. Risk factors for age-related macular degeneration: Pooled findings from three continents. (2001) Ophthalmology 108, 697-704. VEGF-A is present in CNV membranes surgically excised from patients with AMD (Lopez, P. F., Sippy, B. D., Lambert, H. M., Thach, A. B. & Hinton, D. R. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes. (1996) Invest Ophthalmol Vis Sci 37, 855-68), and pharmacological inhibition of VEGF-A decreases experimental laser-induced CNV. Saishin, Y. et al. VEGF-TRAPR1R2 suppresses choroidal neovascularization and VEGF-induced breakdown of the blood-retinal barrier. (2003) J Cell Physiol 195, 241-8. These data are the bases for current clinical trials of anti-VEGF-A therapy in patients with AMD.
However, the precise role of VEGF-A in CNV still is unclear. While subretinal injection of viral vectors coding for VEGF-A leads to retinal pigmented epithelium (RPE) overexpression of VEGF-A and subsequent CNV (Spilsbury, K., Garrett, K. L., Shen, W. Y., Constable, I. J. & Rakoczy, P. E. Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization. (2000) Am J Pathol 157, 135-44), transgenic VEGF-A upregulation directed by RPE-specific promoters such as RPE65 or VMD2 does not produce CNV. Schwesinger, C. et al. Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinal pigment epithelium. (2001) Am J Pathol 158, 1161-1172. Oshima, Y. et al. Increased expression of VEGF in retinal pigmented epithelial cells is not sufficient to cause choroidal neovascularization. J Cell Physiol Published Online: 7 Jun. 2004, DOI: 10.1002/jcp.20110 (2004). However, transgenic VEGF-A upregulation coupled with subretinal injection of null viral vector induced CNV (Oshima, Y. et al. 2004), suggesting that increased VEGF-A alone is insufficient to induce CNV without coexisting mechanical trauma or immune deviation. In addition, the results of a large clinical trial of an anti-VEGF-A aptamer in CNV have yielded mixed results (http://www.fda.gov/ohrms/dockets/ac/04/briefing/2004-4053B1—02_FDA-Backgrounder.pdf).
This trial demonstrated an inverse dose response, with the highest dose of Macugen® (Eyetech Pharmaceuticals, Inc.) showing no significant treatment effect. Although the lowest dose decreased the rate of vision loss over 1 year, it did not alter the inexorable increase in CNV lesion size. In addition, because a large fraction of patients treated with Macugen® also received photodynamic therapy with verteprofin (Visudyne®, QLT, Inc. and Novartis Opthalmics), a currently approved and widely used treatment, it is difficult to extract the effect of Macugen®. Paradoxically Visudyne® reduces the rate of increase in CNV lesion size despite increasing VEGF-A expression in the choroid. Schmidt-Erfurth, U. et al. Influence of photodynamic therapy on expression of vascular endothelial growth factor (VEGF), VEGF receptor 3, and pigment epithelium-derived factor. (2003) Invest. Ophthalmol. Vis. Sci. 44, 4473-4480.
While the role of VEGF-A in CNV is still unresolved, recruitment of macrophages, which is spatiotemporally correlated with arborizing CNV in patients with AMD, is known to be operative in the development of CNV. Grossniklaus, H. E. et al. Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization. (2002) Mol Vis 8, 119-26. Sakurai, E. et al. Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization. (2003) Invest Ophthalmol Vis Sci 44, 2743-9. Sakurai, E., Anand, A., Ambati, B. K., van Rooijen, N. & Ambati, J. Macrophage depletion inhibits experimental choroidal neovascularization. (2003) Invest Ophthalmol Vis Sci 44, 3578-85. Because VEGF-A is a chemoattractant for monocyte-derived cells, there is a need for understanding the interactions between VEGF-A and macrophages in CNV, and the roles of VEGFR-1 and VEGFR-2 in this process.
Tight regulation of VEGF-A following injury permits coordinated orchestration of angiogenesis and inflammation, initiated by arrival of inflammatory cells followed by endothelial proliferation. The transient decline in SPARC immediately following injury temporarily unsilences VEGFR-1 tyrosine kinase activity, promoting VEGF-A signaling via VEGFR-1. Increasing VEGF-A levels during this period not only disrupts leukocyte recruitment by inducing anti-inflammatory pathways via excess VEGFR-1 stimulation, but also arrests endothelial cells, disadvantaging their proliferation in the angiogenesis stage. This Janus-like effect reveals novel therapeutic strategies to modulate angiogenesis in the setting of inflammation and highlights the importance of developing the ability to assay expression of markers such as SPARC to target therapeutics more specifically.